The present invention relates to novel peptides being epitopes of the human 60 KDa heat shock protein (hsp60) and to pharmaceutical compositions comprising them for the diagnosis and treatment of insulin-dependent diabetes mellitus (IDDM).
Type I diabetes, or IDDM, is an autoimmune disease caused by T cells that attack and destroy the insulin-producing xcex2-cells located in the islets of the pancreas (Castano and Eisenbarth, 1990). The autoimmune process culminating in IDDM begins and progresses without symptoms. The disease surfaces clinically only when the cumulative loss of xcex2-cells exceeds the capacity of the residual xcex2-cells to supply insulin. Indeed, the collapse of glucose homeostasis and clinical IDDM is thought to occur only after 80-90% of the xcex2-cells have been inactivated by the immune system. Thus, patients who can be identified as suffering from IDDM are bound to be in an advanced stage of autoimmune destruction of their xcex2-cells. Moreover, diagnosis of incipient, pre-clinical diabetes by the detection of immunological markers of xcex2-cell autoimmunity can be made only after the onset of the autoimmune process. Therefore, the therapeutic quest is to find a safe, specific and effective way to turn off an autoimmune process that is already well underway.
The present inventors have examined this question before by studying the spontaneous diabetes developing in mice of the NOD strain, which is considered to be a faithful model of human IDDM (Castano and Eisenbarth, 1990). NOD mice develop insulitis around 4 weeks of age, which begins as a mild peri-islet infiltrate and progresses to severe intra-islet inflammation. Hyperglycemia, which attests to insulin insufficiency, begins in the females in our colony at about 14-17 weeks of age. Bad 35-40 weeks of age, almost all the female NOD mice have developed severe diabetes and most die in the absence of insulin treatment. Male NOD mice have a lower incidence of disease, for reasons that are not clear. The diabetes of NOD mice has been shown to be caused by autoimmune T cells (Bendelac et al., 1987).
T cell reactivity and autoantibodies to various antigens have been detected in human IDDM patients as well as in NOD mice (Elias, 1994), and it is not clear whether immunity to any single one of the possible target antigens is the primary cause of the disease. Beyond the question of causation is the question of therapy.
It has been demonstrated that the initiation of the autoimmune process in NOD mice can be prevented by subjecting the mice, before the onset of diabetes, to various manipulations such as restricted diet, viral infections, or non-specific stimulation of the immune system (Bowman et al., 1994). NOD diabetes is also preventable by induction of immunological tolerance in pre-diabetic mice to the antigen glutamic acid decarboxylase (Kaufman et al., 1993; Tisch et al., 1993).
Insulin dependent diabetes mellitus (IDDM) developing spontaneously in NOD female mice has been associated with immune reactivity to a variety of self-antigens (Bach, 1994). Notable among these antigens is the p277 peptide from the sequence of the mammalian 60 kDa heat shock protein (hsp60) molecule. This corresponds to residues 437-460 in the human hsp60 molecule (Elias et al 1991, Israel Patent Application No. 94241, PCT patent publication WO90/10449). The human p277 peptide has the following sequence:
Val-Leu-Gly-Gly-Gly-Cys-Ala-Leu-Leu-Arg-Cys-Ile-Pro-Ala-Leu-Asp-Ser-Leu-Thr-Pro-Ala-Asn-Glu-Asp (a.a. 437-460 of SEQ ID NO:1).
Pre-diabetic NOD mice manifest spontaneous, diabetogenic T cell responses to hsp60 and to the human (2) or mouse variants of the p277 peptide (3). The mouse and human peptides differ by 1 amino acid and are immunologicaly cross-reactive (3). Some non-diadetes prone strains of mice, such as C57BL/6, develop transient hyperglycemia and insulitis when immunized to p277 covalently conjugated to a foreign immunogenic carrier molecule (4). And mice of the C57BL/KsJ strain develop spontaneous T-cell responses to hsp60 and to p277 after treatment with a very low dose of the xcex2-cell toxin streptozotocin (STZ) that induces autoimmune diabetes (5).
In addition to being involved in the expression of the disease, peptide p277 appears to be functional in healing the autoimmune process: Subcutaneous administration of p277 in incomplete Freund adjuvant (IFA; mineral oil) led to arrest of disease progression in young NOD mice (2) or in 12-17 week old NOD mice with advanced insulitis (6, 7). Both the human (6, 7) and mouse (3) variants of p277 were effective. NOD mice transgenic for the mouse hsp60 gene on an MHC class II promoter showed down-regulation of their spontaneous T-cell proliferative response to p277 and a significant proportion of the mice were spared the development of diabetes (8). Moreover, administration of p277 to C57BL/KsJ mice aborted the development of autoimmune diabetes in mice that had received earlier a very low dose of STZ; treatment of these mice with a peptide of the GAD65 molecule was not effective (9).
Variants of the p277 peptide in which one or both cysteine residues at positions 6 and 11 were replaced by valine residues, designated as p277(Val6), p277(Val11) and p277(Val6-Val11), respectively, were described in corresponding Israel Patent Application No. 112094, and shown to be as active as p277 in the treatment of diabetes.
It is an object of the present invention to provide additional peptides of human hsp60, such peptides being useful for diagnosis and treatment of IDDM.
In a study of fragments and peptides of the human hsp60 molecule, it was unexpectedly found that IDDM patients and NOD mice are responsive to other hsp60 T-cell epitopes that may be used for diagnosis and therapy of IDDM. These epitopes, by themselves or in conjunction with p277 or a p277 variant selected from p277(Val6), p277(Val11) and p277(Val6-Val11), may improve the efficacy of the treatment.
These new peptides are identified in Table 1.
Other peptides of hsp60, SEQ ID NO:1, including those designated p278 (corresponding to positions 458-474 of SEQ ID NO:1 in the human hsp60 sequence), p19 (corresponding to positions 271-290 of SEQ ID NO:1 in the human hsp60 sequence), and p21 (corresponding to positions 301-320 of SEQ ID NO:1 in the human hsp60 sequence were shown not to be as effective. It is noted that the amino terminus of p278 overlaps with the effective p277 peptide by three residues (NED) and the carboxy terminus of p278 overlaps with the effective p32 peptide by residues (EIIKRTLKI). Thus the remaining 11 residues of p32 are critical (PAMTIAKNAGV).
The present invention thus relates to the peptides identified in Table 1, and salts and functional derivatives thereof.
It is further an object of the present invention to provide methods and kits for the early diagnosis of IDDM using the peptides of the invention. In the course of developing IDDM, animals express hsp60 molecules, or molecules which are cross-reactive therewith, which find their way into the blood and urine of the animals. They also express antibodies and T cells directed specifically to such molecules. Thus, the presence of hsp60 (or molecules which are cross-reactive therewith) or antibodies or T cells specific thereto in blood or urine, serves as an assay for the detection of the IDDM process before the destruction of beta cells is completed and the individual is doomed to life-long diabetes.
The presence or incipience of IDDM in a patient can be diagnosed by testing the blood or urine of said patient for the presence of antibodies or T cells which are immunologically reactive with human hsp60, using as antigen a peptide p12 or p32 of the invention.
Accordingly, the present invention provides a method for diagnosing the presence or incipience of IDDM in a patient, comprising testing said patient for the presence of anti-hsp60 antibodies or of a T cell which immunoreacts with hsp60 using a peptide of the present invention as antigen, whereby a result indicating the positive presence of anti-hsp60 antibodies or of a T cell which immunoreacts with hsp60, indicates a high probability of the presence or incipience of IDDM.
In the method for diagnosing IDDM, the patient may be tested for the presence of anti-hsp60 antibodies, wherein said test method may comprise a radioimmunoassay or an ELISA test.
The patient may also be tested for the presence of a T cell which immunoreacts with hsp60. In one embodiment of this aspect, the test method comprises a T cell proliferation test comprising the steps:
(i) preparing a mononuclear cell fraction containing T cells from a blood sample obtained from said patient;
(ii) adding to said mononuclear cell fraction an antigen selected from the pepdtide of the invention;
(iii) incubating said cell fraction in the presence of said antigen for a suitable period of time and under suitable culture conditions;
(iv) adding a labeled nucleotide to the incubated cell culture of (iii) at a suitable time before the end of said incubation period to provide for the incorporation of said labeled nucleotide into the DNA of proliferating T cells; and
(v) determining the amount of proliferating T cells by analysis of the amount of labeled nucleotide incorporated into said T cells.
In step (iv) above, said labeled nucleotide is preferably 3H-thymidine. The determination of the amount of proliferating T cells is made by calculation of the stimulation index of the T cells by standard methods.
In another embodiment of this aspect of the invention, the test method comprises a T-cell cytokine response test, in which steps (i) to (iii) are as in the above T cell proliferation test, and in a fourth step (iv) the presence of cytokine, such as IFN-xcex3, IL-2, IL-4, IL-6, IL-10, TNFxcex1 or TGFxcex2, secreted by the responding lymphocytes into the medium, is detected by standard methods with commercially available kits.
In another aspect, the invention provides an in vivo method wherein an antigen selected from the peptides of the invention is injected subcutaneously into a patient and the occurrence of a detectable skin reaction (delayed type hypersensitivity; DTH) is observed.
The present invention also relates to means for performing such assays, as well as kits for performing such assays. The kits may be prepared for carrying out any of the various assays used for accomplishing the present invention. Each such kit includes all of the materials necessary to conduct a single assay or a fixed number of assays. For example, such a kit for determining the presence of anti-hsp60 antibodies may contain a solid-phase immobilized peptide of the invention and a tagged antibody capable of recognizing the non-variable region of the anti-hsp60 antibody to be detected, such as tagged anti-human Fab. The kit may also contain directions for using the kit and containers to hold the materials of the kit. Any conventional tag or label may be used, such as a radioisotope, an enzyme, a chromophore or a fluorophore. A typical radioisotope is iodine-125 or sulfur-35. Typical enzymes for this purpose include horseradish peroxidase, horseradish galactosidase and alkaline phosphatase.
A kit for diagnosing the presence of IDDM by testing for the presence of anti-hsp60 antibodies, comprises:
(i) an antigen selected from the peptides of the invention; and
(ii) a tagged antibody capable of recognizing the non-variable region of said anti-hsp60 antibodies to be detected.
A kit for diagnosing the presence of IDDM by testing for the presence of a T cell which immunoreacts with hsp60, will comprise:
(i) an antigen selected from the peptides of the invention;
(ii) a suitable medium for culture of lymphocytes (T cells); and
(iii) either a labeled nucleotide for the T cell proliferation test, or a cytokine, e.g., interferon-gamma, assay kit, for the cytokine test.
For the in vivo test, the kit will comprise only a peptide of the invention in a suitable form for injection.
The present invention further relates to means for preventing or treating IDDM. Vaccination with an antigen peptide of the present invention can provide a specific down regulation of autoimmunity to the antigen, and effectively creates a resistance to the autoimmune process of IDDM. The same is true with respect to vaccination with T cells specific to such antigens, in attenuated or avirulent form or after having been treated to improve their antigenicity, or fragments or active fractions thereof. If the patient is shown to already be in the pre-clinical incipient stages of IDDM, injection with such an antigen or T cell (or fraction) can create a down regulation of autoimmunity for this antigen and thus arrest the autoimmune process before significant, permanent damage is done. The peptide can also be used as a therapeutic agent to arrest the autoimmune process even after it is far advanced, as shown recently by the laboratory of the present inventors regarding the treatment of NOD mice with the peptide p277(Elias and Cohen, 1994).
Accordingly, the present invention provides a preparation for preventing or treating insulin-dependent diabetes mellitus (IDDM), comprising: (a) T cells which have developed specificity for a protein or peptide which is immunologically cross-reactive with a peptide of the invention, which cells have been activated by incubating in the presence of said peptide; (b) said T cells which have been irradiated or otherwise attenuated; (c) said T cells which have been subjected to pressure treatment by means of hydrostatic pressure, treatment with a chemical cross-linking agent and/or treatment with a cytoskeletal cross-linking agent; (d) fragments of or surface proteins shed from (a), (b) or (c); or (e) a peptide consisting essentially of the variable region of the receptor of (a) specific for said protein, or a salt, functional derivative, precursor or active fraction thereof.
In a preferred embodiment of the invention, the preparation comprises human T cells that have developed specificity by in vitro contact with said peptide of the invention.
The present invention also provides a pharmaceutical composition for the prevention or treatment of IDDM comprising a pharmaceutically acceptable carrier and, as active principle, an effective amount of a peptide of the invention, a salt or a functional derivative thereof.
The invention further relates to a method of preventing or treating IDDM which comprises administering to a patient in need thereof a pharmaceutical composition comprising a peptide of the invention, a salt or a functional derivative thereof, or a preparation comprising T cells which have developed specificity to said peptide of the invention.